Energy is the foundation of human society development. Facing the global energy crisis, the oil remains in short supply. Bio-energy has become a search focus due to its properties of renewability, and with the unique advantages of no need of food, land and forage, microalgae biodiesel has been attracting increasing attention around the world.
Different species of microalgae have various ability to synthesize triacylglycerol (TAG). It has been revealed that the eukaryotic microalgae (such as green algae or diatom etc.) has higher oil content, which is a promising alga species to produce biodiesel. Myrmecia incisa Reisigl H4301 is a single-cell freshwater microalga belonging to Chlorophyta with accumulated amounts of TAG, especially under a nitrogen starvation stress. Therefore, Myrmecia incisa can be regarded as a potential algal species for production of biodiesel.
TAG is the major storage form of lipid in plants, and plays an important role in the growth and development of plants. Three TAG synthesizing enzymes have been found: diacylglycerol acyltransferase (DGAT, EC 2.3.1.20), phospholipid: diacylglycerol acyltransferase (PDAT, EC 2.3.1.158), and diacylglycerol transacylase (DGTA). Therein, DGAT, the most important enzyme in TAG synthesis, catalyzes the final step in TAG biosynthesis along Kennedy pathway and is also the only rate-limiting enzyme of this pathway. To date, three DGAT families have been discovered, which are DGAT1, DGAT2 and DGAT3.
A Chinese Paper was published which was titled “the transcriptome sequencing and function annotation of lipid metabolism-related genes in the green microalga Myrmecia incisa” in the Journal of Shanghai Ocean University, volume 21, NO. 5, September 2012. In order to give a deep insight of the metabolic process of arachidonic acid and lipid in Myrmecia incisa, high through-put transcriptome pyrosequencing of Myrmecia incisa was conducted using the sequencer Roche 454 GS FLX, and a total of 382 468 high quality reads were obtained, which accounts for 97.14% of the original reads, with an average length of 322 nucleotides totally 123 Mb. After clustering and assembly using the CAP3 software, these reads were assembled into 22 714 contigs and 25 621 singletons. Homology search, comparison and annotation and classification of gene function were performed against public data bases, and the lipid metabolism pathway in Myrmecia incisa Reisigl was established based on the annotated genes in the transcriptome. But this paper does not disclose Myrmecia incisa Reisigl diacylglycerol acyltransferase cDNA sequence, and it is recognized by those skilled in the art that the method of high-throughput sequencing is not 100% accurate and is highly possible for inaccuracy, thus according to the paper, a person of ordinary skill in the art is difficult to obtain the accurate gene sequence of Myrmecia incisa Reisigl diacylglycerol acyltransferase.
Chinese patent document CN201210477507.7 whose date of publication is Feb. 27, 2013, titled “a DNA sequence coding Myrmecia incisa Reisigl diacylglycerol acyltransferase and the use thereof shows that based on Myrmecia incisa Reisigl transcriptome sequencing data, a full-length cDNA sequence of a Myrmecia incisa Reisigl diacylglycerol acyltransferase (MiDGAT2) gene was obtained by screening. Homology alignment analysis indicated that the Myrmecia incisa Reisigl diacylglycerol acyltransferase gene was of the DAGT2-gene family. Further obtained was the full-length DNA sequence of MiDGAT2. Heterologous expression in a TAG synthesis defective strain H1246 of yeast, proved that the protein encoded by the gene has the capability of triacylglycerol synthesis. However, Myrmecia incisa Reisigl is a eukaryotic alga, and it may contain more than one diacylglycerol acyltransferase, and TAG synthesis may be catalyzed by multiple isozymes. An isozyme/isoenzyme refers to an enzyme that has different properties (different Vmax and/or Km) but catalyzes the same reaction, which is also called isofunctional enzymes. The amount of isoenzymes may vary between different organs and tissues in an organism, and the isoenzymes may also appear in different organelles of any eukaryotic cells. Their differences can be shown in the primary and quaternary structure of protein, or in the process after translation. Their enzymatic activity can be regulated in response to specific physiological conditions in the cell. Isoenzyme is the result of gene variation; while gene variation is the molecular evolution during the evolution process in order to adapt to the increasingly complex metabolism. Gene differentiation facilities the adaptation of different metabolic needs in different tissues or different organelles, and is the protein phenotype of gene coding. Therefore, finding and investigating Myrmecia incisa Reisigl diacylglycerol acyltransferase gene sequence is of great importance to the study of Myrmecia incisa Reisigl evolution, genetic variation and provides a new method of synthesizing TAG by genetic engineering.